Interpretive Summary: The nutritional and physical properties of vegetable oils are determined by their fatty acid composition, and the various fatty acid compositions found in vegetable oils are generated by the action of enzymes called fatty acid desaturases. Despite their importance in determining fatty acid composition of oils, very little is known about how these enzymes actually operate in plant cells. In collaboration with scientists at the University of Guelph, McMaster University, and Rice University, scientists at SRRC have determined how these enzymes are organized in specific locations within plant cells to carry out their functions. Information learned during these studies revealed new information about many other proteins that are also located in a similar region of the cell. Therefore, the results presented in this manuscript not only shed substantial light on the functioning of plant fatty acid desaturases, but also has a much broader impact on many other areas of plant science research. This information will have an immediate impact on scientists studying basic aspects of fatty acid production, but will also be beneficial to scientists who are interested in using fatty acid desaturases to produce desired amounts of specific fatty acids in transgenic crops.

Technical Abstract:
Fatty acid desaturases (FADs) play a prominent role in plant lipid metabolism and are located in various subcellular compartments including the endoplasmic reticulum (ER). To investigate the biogenesis of ER-localized membrane-bound FADs, we characterized the mechanisms responsible for insertion of Arabidopsis FAD2 and Brassica FAD3 into ER membranes and determined the molecular signals that maintain their ER residency. Using in vitro transcription/translation reactions with ER-derived microsomes we show that both FAD2 and FAD3 are efficiently integrated into membranes by a co-translational, translocon-mediated pathway. We also demonstrate that while the C terminus of FAD3 (-KSKIN) contains a functional prototypic dilysine ER retrieval motif, FAD2 contains a novel C-terminal aromatic amino acid-containing sequence (-YNNKL) that is both necessary and sufficient for maintaining localization in the ER. Co-expression of a membrane-bound reporter protein containing the FAD2 C terminus with a dominant-negative mutant of Arf1 abolished transient localization of the reporter protein in the Golgi, indicating that the FAD2 peptide signal acts as an ER retrieval motif. Mutational analysis of the FAD2 ER retrieval signal revealed a sequence-specific motif consisting of Phi-X-X-K/R/D/E-Phi-COOH, where Phi are large hydrophobic amino acid residues. Interestingly, this aromatic motif was present on a variety of other known and putative ER membrane proteins including cytochrome P450 and the peroxisomal biogenesis factor Pex10p. Taken together, these data describe the insertion and retrieval mechanisms of FADs and define a new ER localization signal in plants that is responsible for the retrieval of escaped membrane proteins back to the ER.